Insulin analogues

Abstract

Novel rapid-acting human insulin analogues are provided having less tendency to self-association into dimers, tetramers, hexamers, or polymers. The novel human insulin analogues are formed by substituting one or more of the amino acid residues of human insulin with naturally occuring amino acid residues. The amino acid residue substitutions are preferably more hydrophilic than the natural amino acid residue at the respective position in the molecule. Furthermore, the insulin analogues have the same charge or a greater negative charge at neutral pH than that of human insulin. Preferred amino acid substitutions are Asp, Glu, Ser, Thr, His, and Ile, and more preferred substitutions are Asp and Glu. The novel insulin analogues can be used for the preparation of rapid-acting insulin solutions.

We claim: 
     
       1. Rapid acting human insulin analogues, characterized in that they have the formula I ##STR6## wherein except as indicated hereinafter the X's are the amino acid residues of human insulin and at least one but not more than 4× is different from the amino acid residue of human insulin at the respective position in the insulin molecule the net function of which substitution(s) is to impart to the molecule the same charges or a greater negative charge at neutral pH than that of human insulin, with the proviso that when X in position B(5) is Ala, or when X in position B(9) is Leu; or when X in position B(10) is Asn or Leu; or when X in position B(12) is Asn; or when X in position B(26) is Ala, then at least one of the remaining X's is different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further proviso that at least one substitution is at an X in the B-chain but X in position B(10) is not Asp. 
     
     
       2. Insulin analogues according to claim 1, wherein the amino acid residue substitutions are more hydrophilic than the amino acid residue of human insulin at the respective position in the insulin molecule. 
     
     
       3. Human insulin analogues according to claim 1, wherein the amino acid substitutions are selected from the group consisting of Glu, Ser, Thr, His, and Ile. 
     
     
       4. Human insulin analogues according to claim 1, wherein the amino acid residue substitution is Glu. 
     
     
       5. Human insulin analogues according to claim 1, wherein at least one X in position B(9), B(10), B(12), B(26), B(27), or B(28) is different from the amino acid residue at the corresponding site in the molecule of human insulin. 
     
     
       6. Injectable solutions with insulin activity, characterized in that they contain a human insulin analogue according to claim 1 or a pharmaceutically acceptable salt thereof in aqueous solution preferably at neutral pH. 
     
     
       7. Human insulin analogues according to claim 1 with at least one B-chain amino acid residue substitution therein from the following list:   ______________________________________                                    
B(9): Asp, Glu, His, Gln or Asn;                                          
B(10): Arg, Glu, Asn, Gln, Ser, Thr, Ile;                                 
B(12): His, Ile, or Tyr;                                                  
B(16): Asp, Glu, Gln, Asn, Ser, Thr or His;                               
B(17): Ser, Thr, Asn, Gln, Glu, Asp, or His;                              
B(20): Ser, Thr, Asn, Gln, Asp, Glu or His;                               
B(26): Asp, Glu, Asn, Gln, Ser, Thr or His;                               
B(27): Asp, Glu or His;                                                   
B(28): Asp, Glu or His.                                                   
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       8. Human insulin analogues according to claim 7, wherein one or more of said substitutions at position B(9), B(10), B(16), B(27) and B(28) are combined with 1 to 3 substituents from the following list:   ______________________________________                                    
A(8): His, Gly, Gln, Glu, Ser, Asn, Pro or Asp;                           
A(13): His, Glu, Asp, Thr, Ser, Asn or Gln;                               
A(21): Asp or Glu;                                                        
B(1): Glu, Asp, Thr or Ser;                                               
B(2): Arg, His Ala, Glu, Asp, Thr, Pro, Gly, Gln, Ser or                  
Asn.                                                                      
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       9. Human insulin analogues according to claim 7, wherein one or more of said substitutions at position B(9), B(17), B(27), or B(28) are combined with one substitution in B(14) selected from the group consisting of Glu, Asp, Asn, Gln, Ser, Thr and Gly. 
     
     
       10. Human insulin analogues according to claim 7, wherein one of said substitutions is combined with one substitution in B(18) selected from the group consisting of Ser, Thr, Asn, Glu and His. 
     
     
       11. Human insulin analogues according to claim 1 wherein from 2-4 residues are different from the amino acid residue of human insulin. 
     
     
       12. Rapid acting human insulin analogues, characterized in that they have the Formula I ##STR7## wherein except as indicated hereinafter the X's are the aamino acid residues of human insulin at least 1 but not more than 4 being different from the amino acid residue of human insulin at the respective position in the insulin molecule, with the proviso when X in position B(9) is Leu; or when X in position B(10) is Asn then at least one of the remaining X's are different from the amino acid residues of human insulin at the respective position in the insulin molecule and with the further provisio that at least one X in the B-chain is different, the net function of which substitution(s) being to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, said substitution(s) being selected from the following:   ______________________________________                                    
Position                                                                  
        Amino acid residue substitutions                                  
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A8      His, Gly, Gln, Glu, Ser, Asn, Asp, Pro                            
A9      Gly, Asp, Glu, Thr, His, Gln, Asn, Ala, Pro                       
A10     Le, Pro, Val, His, Ala, Glu, Asp, Thr, Gln,                       
        Asn                                                               
A13     Pro, Val, Arg, His, Ala, Glu, Asp, Thr, Gly, Gln,                 
        Asn, Asp, Ser, Thr                                                
A21     Asp, Glu, Ser, Thr                                                
B1      Glu, Asp, Thr, Ser, Gly                                           
B2      Arg, His, Ala, Glu, Asp, Thr, Pro, Gly, Gln, Ser,                 
        Asn                                                               
B5      Glu, Asp, Thr, Ser, Gln, Asn                                      
B9      Asp, Pro, Glu, Ile, Leu, Val, His, Thr, Gln, Asn,                 
        Met, Tyr, Trp, Phe                                                
B10     Arg, Glu, Asn, Gln, Ser, Thr                                      
B12     Ile, Tyr                                                          
B14     Glu, Asp, Asn, Gln, Ser, Thr, Gly                                 
B16     Asp, Glu, Gln, Asn, Ser, Thr, His, Arg                            
B17     Ser, Thr, Asn, Gln, Glu, Asp, His                                 
B18     Ser, Thr, Asn, Gln, His                                           
B20     Gln, Ser, Asn, Asp, Glu, Arg, Thr                                 
B26     Asp, Glu, Ser, Thr                                                
B27     Asp, Glu                                                          
B28     Asp, Glu.                                                         
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       13. Human insulin analogues selected from the group consisting of B(27) Glu human insulin, B(12) Ile human insulin, B(12) Tyr human insulin, A(21) Asp B(27) Gtu human insulin, B(9) Asp human insulin, A(21) Asp B(9) Asp B(27) Glu human insulin, B(9) Asp B(27) Glu human insulin, and B(28) Asp human insulin, said analogues being characterized by rapid acting insulin activity. 
     
     
       14. A human insulin analogue according to claim 13, wherein the human insulin analogue is B(28) Asp human insulin.